BS13 Cross talk between the PKCα and TGF-β signalling pathways during vascular calcification

Vascular calcification is an independent risk factor for mortality in chronic kidney disease. We reported previously that knocking-down protein kinase Cα (PKCα) increased vascular calcification in vitro and in vivo, and crucially, PKCα may exert these effects by increasing pro-calcific transforming growth factor-β (TGF-β) signalling in vascular smooth muscle cells (VSMCs). Previous studies have shown that activating PKC with phorbol 12-myristate 13-acetate increases ERK phosphorylation in LX-2 cells, and ERK can regulate the phosphorylation of SMAD2. Therefore, this study tests the hypothesis that PKCα regulates VSMC calcification via ERK-TGF-β signalling. In vitro, TGF-β1-induced ERK phosphorylation was increased in VSMCs treated with the PKCα activator, prostratin (P<0.05). Prostratin decreased TGF-β1-induced SMAD2 phosphorylation in VSMCs (P<0.05), and this decrease in SMAD2 phosphorylation was prevented by co-treating the cells with the ERK inhibitor, U0126. In contrast, TGF-β1-induced ERK phosphorylation was reduced in PKCα siRNA-treated VSMCs (P<0.05). PKCα knock-down increased TGF-β1-induced SMAD2 phosphorylation (P<0.05), and this increase in SMAD2 phosphorylation was prevented by co-treating the cells with the ERK activator, tert-butylhydroquinone (TBHQ). To further define the link between PKCα and ERK activation in VSMCs, control siRNA- and PKCα siRNA-transfected VSMCs were cultured with vehicle or TBHQ in the presence of β-glycerophosphate (β-GP). Knocking-down PKCα expression with siRNA significantly increased β-GP-induced VSMC mineralisation when compared to controls (P<0.05), and this increase in mineralisation was prevented by co-treating VSMCs with TBHQ. To analyse the relationship between PKCα and SMAD2 phosphorylation during calcification in vivo, immunohistochemistry was performed. PKCα and phosphorylated SMAD2 immunostaining localised to areas directly adjacent to, and within, calcified regions of human atherosclerotic plaques. Uraemia-induced calcification was increased in the aortic arch of PKCα−/− mice, and this was associated with increased phosphorylated SMAD2 immunostaining in the aortic arch of PKCα−/− mice when compared to wild-type controls (P<0.05). In conclusion, these data suggest that PKCα may regulate TGF-β1-induced SMAD2 phosphorylation via ERK activation in VSMCs. The PKCα-ERK-TGF-β signalling axis could represent a new area of research for vascular calcification. Conflict of Interest None