Objective      To down-regulate the expression of prolein-rich tyrosine kinase 2 (Pyk2) in cultured adult rat cardiac fibroblasts (CFs) with short hairpin RNA interference

and evaluate the role of Pyk2/PI3K/NF-κB in cell phenotypic transition and migration.     Methods      The Pavu6＋27-Pyk2 shRNA expression vector was constructed by gene recombination

then transfected into cultured adult rat CFs stimulated by angiotension Ⅱ. Untransfected cells (control)

cells transfected with Pavu6＋27 vector (empty vector) and those transfected with negative shRNA vector (negative control) were used as control. Transwell chamber test and in vitro scratching assay were adopted to evaluate the cell migration. Change of α-smooth muscle actin (α-SMA) in CFs was analyzed in transwell chamber by immunofluorescence. The protein expressions of Pyk2 and phosphate phosphoinositide-3-kinase protein (PI3K) p85 subunit in CFs were determined by Western blotting. Electrophoretic mobility shift assay was employed to determine the activation of NF-κB.     Results      After Pyk2 shRNA transfection

transwell assay showed the number of migrated CFs was increased with the prolongation of culture. However

comparing to control group

the number of migrated CFs in Pyk2 shRNA transfection group was deceased significantly after transfection (19.1±6.1 vs 30.8±8.6 at 6 h

37.7±12.4 vs 53.6±11.3 at 12 h

78.9±18.5 vs 102.3±21.7 at 24 h

P0.05

0.01). But no difference was found among these control

empty vector and negative control. Immunofluorescence staining showed that α-actin expression and migration in CFs was inhibited after Pyk2 shRNA transfection. In vitro scratching assay revealed there were significant difference between the migration distance of CFs with Pyk2 shRNA transfection and control (0.838 vs 1.213 mm at 24 h

1.626 vs 2.049 mm at 48 h

P0.05

0.01).  The protein expression of Pyk2 (0.47 vs 0.61) and phosphated PI3K p85 (0.31 vs 0.42) were declined after Pyk2 shRNA transfection (P0.05). Electrophoretic mobility shift assay demonstrated that the NF-κB DNA binding activity was reduced (P0.05).     Conclusion      Pyk2/PI3K/NF-κB signal pathway might be critical in mediating CFs migration and phenotypic transition

Pyk2 is a promising therapeutic candidate in myocardial fibrosis.