Aim: To investigate the role of Ca(2+)-calmodulin dependent protein kinase (CCDPK) on basic fibroblast growth factor (bFGF)-induced vascular smooth muscle cell (VSMC) proliferation and the inhibitory effect of antisense CCDPK oligonucleotides (ODN).
Methods: Before being exposed to bFGF, cultured rat VSMC CCDPK activity was inhibited by pretreatment with either a phosphorothioate-protected 17-mer antisense CCDPK ODN-directed against the initiation of translation sites of the p42 and p44 CCDPK isoform or with CCDPK kinase inhibitor PD98059. All ODN were introduced into cells by liposomal transfection. DNA synthesis was measured by [3H]thymidine incorporation. P44- and p42-CCDPK protein expression and phosphorylation were measured by Western blot.
Results: PD98059 inhibited bFGF-induced phosphorylation of CCDPK and DNA synthesis. Antisense CCDPK ODN 0.2-0.8 mumol.L-1 reduced both p44- and p42-CCDPK expression and phosphorylation of CCDPK in a concentration-dependent manner and DNA synthesis induced by bFGF. Lipofectin alone or sense and random CCDPK ODN did not affect p44- and p42-CCDPK protein expression or bFGF-induced phosphorylation of CCDPK or DNA synthesis.
Conclusion: bFGF-stimulated rat VSMC proliferation is mediated by CCDPK. The antisense CCDPK ODN can inhibit bFGF-induced VSMC proliferation through down-regulating p44- and p42-CCDPK level.