PKCα is involved in the migration of SW620cells promoted by VIIa/TF-activated PAR2

Objective To explore the possible mechanism of protein kinase C α(PKCα) involved in the migration of SW620 cells promoted by Ⅶa/tissue factor(TF) activated protease-activated receptor 2(PAR2).Methods After SW620 cells were treated by PAR2 agonist(PAR2-AP),coagulation factor Ⅶa,PKCagonist(PMA),anti-TF antibody(α-TF),anti-PAR2 antibody(α-PAR2) and isotype control antibody(mopc-21) respectively,the expressions of PKCα and p-PKCα in SW620 cells were detected by Western blot,and the distribution of PKCα by immunofluorescence assay.After SW620 cells were pretreated with PMA(100 nmol/L),factor Ⅶa(10 nmol/L) and PKCα inhibitor(safingol,10 μmol/L) respectively,the migration of SW620 cells was detected by Transwell test,and the expression level of MMP-9 mRNA by quantitative real-time PCR(q-PCR).Results PMA(100 nmol/L),factor Ⅶa(10 nmol/L) and PAR2-AP(100 μmol/L) were able to increase the phosphorylation of PKCα obviously(P0.05),but did not show significant effect on the expression of PKCα(P0.05).Immunofluorescence assay indicated the translocation of PKCα from cytoplasm into nuclear membrane and nucleus of SW620 cells.In the meanwhile,both α-TF and α-PAR2 were able to obviously inhibit the activation of PKCα by factor Ⅶa,while mopc-21 did not show the inhibitory effect.In addition,Transwell test and q-PCR demonstrated that safingol could obviously block the migration of SW620 cells and the expression of MMP-9 mRNA in SW620 cells caused by factor Ⅶa.Conclusion Factor Ⅶa could activate PAR2 based on TF,then upregulate the expression of MMP-9 and promote the migration of SW620 cells via the action of PKCα.