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PAR-3 knockdown enhances adhesion rate of PANC-1 cells via increased expression of integrinαv and E-cadherin.

Segal L, Katz LS, Shapira H, Sandbank J, Geras-Raaka E, Gershengorn MC, Oron Y - PLoS ONE (2014)

Bottom Line: ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells.However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD.PAR-3 KD cells also expressed more E-cadherin mRNA and protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel.

ABSTRACT
The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.

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The effects of PARs knockdown on wound closure on wound closure kinetics.Vector control and the desired PARs knockdown cells were seeded at approximately 80% density. The kinetics of wound closure were performed as described in Methods. The results represent mean±SE of 5 independent experiments performed in triplicates. P<0.05 for Vector vs. PAR-3 KD and >0.05 vs. PAR-1 KD.
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pone-0093879-g003: The effects of PARs knockdown on wound closure on wound closure kinetics.Vector control and the desired PARs knockdown cells were seeded at approximately 80% density. The kinetics of wound closure were performed as described in Methods. The results represent mean±SE of 5 independent experiments performed in triplicates. P<0.05 for Vector vs. PAR-3 KD and >0.05 vs. PAR-1 KD.

Mentions: To examine the adhesion properties to a more physiological substrate, we tested the adhesion of PAR-1 or PAR-3 KDs and vector-control cells to plasticware coated with 200 μg/ml Matrigel. The kinetics of adhesion of vector-control cells was approximately twice faster than to non-coated plastic. PAR-1 KDs, PAR-3 KDs, however, adhered more rapidly, reaching 40% adhesion at 5 min incubation (Fig. 1B). The differences in adhesion rates could be clearly attributed to the initial, rapid adhesion (Fig. 2). The enhanced rate of adhesion of PAR-3 KDs was also reflected in a modest decrease of two-dimensional migration in an in vitro “wound closure” assay, while the rate of PAR-1 KDs was the same as that of vector-control cells (Fig. 3).


PAR-3 knockdown enhances adhesion rate of PANC-1 cells via increased expression of integrinαv and E-cadherin.

Segal L, Katz LS, Shapira H, Sandbank J, Geras-Raaka E, Gershengorn MC, Oron Y - PLoS ONE (2014)

The effects of PARs knockdown on wound closure on wound closure kinetics.Vector control and the desired PARs knockdown cells were seeded at approximately 80% density. The kinetics of wound closure were performed as described in Methods. The results represent mean±SE of 5 independent experiments performed in triplicates. P<0.05 for Vector vs. PAR-3 KD and >0.05 vs. PAR-1 KD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3974847&req=5

pone-0093879-g003: The effects of PARs knockdown on wound closure on wound closure kinetics.Vector control and the desired PARs knockdown cells were seeded at approximately 80% density. The kinetics of wound closure were performed as described in Methods. The results represent mean±SE of 5 independent experiments performed in triplicates. P<0.05 for Vector vs. PAR-3 KD and >0.05 vs. PAR-1 KD.
Mentions: To examine the adhesion properties to a more physiological substrate, we tested the adhesion of PAR-1 or PAR-3 KDs and vector-control cells to plasticware coated with 200 μg/ml Matrigel. The kinetics of adhesion of vector-control cells was approximately twice faster than to non-coated plastic. PAR-1 KDs, PAR-3 KDs, however, adhered more rapidly, reaching 40% adhesion at 5 min incubation (Fig. 1B). The differences in adhesion rates could be clearly attributed to the initial, rapid adhesion (Fig. 2). The enhanced rate of adhesion of PAR-3 KDs was also reflected in a modest decrease of two-dimensional migration in an in vitro “wound closure” assay, while the rate of PAR-1 KDs was the same as that of vector-control cells (Fig. 3).

Bottom Line: ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells.However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD.PAR-3 KD cells also expressed more E-cadherin mRNA and protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel.

ABSTRACT
The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.

Show MeSH
Related in: MedlinePlus