Role of protein kinase C in pitavastatin-induced human paraoxonase I expression in Huh7 cells

We have demonstrated that pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, enhanced human serum paraoxonase (PON1) gene promoter activity and that protein kinase C (PKC) activated PON1 expression through Sp1 in cultured HepG2 cells. We investigated whether PKC was involved in pitavastatin-induced PON1 expression. PON1 gene promoter activity was assessed by a reporter gene assay using cultured Huh7 cells. PON1 protein expression and PKC activation were measured by Western blotting. The binding activity of Sp1 to the PON1 gene upstream was analyzed by electrophoretic mobility shift assay. Both PON1 gene promoter activity and PON1 protein expression were elevated by pitavastatin stimulation. The effects of pitavastatin on PON1 promoter activity and PON1 protein expression were attenuated by both bisindolylmaleimide IX (Ro-31-8220) and bisindolylmaleimide I. Electrophoretic mobility shift assay showed that pitavastatin increased the Sp1-PON1 DNA binding, and this effect was attenuated by Ro-31-8220. Pitavastatin activated atypical PKC, but never conventional or novel PKC. Myristoylated pseudosubstrate peptide inhibitor of PKCζ abolished the pitavastatin-increased PON1 promoter activity; however, calphostin C and Gö6976 (PKC inhibitors except for PKCζ) did not influence the promoter activity. In addition, an overexpression of dominant negative form of PKCζ expression vector obviously decreased pitavastatin-induced PON1 promoter activation. These observations suggest that pitavastatin activates PKC, especially PKCζ isoform, which increases the binding intensity of Sp1 to PON1 DNA promoter responsible for enhanced transcription of PON1 gene and increased PON1 protein expression in Huh7 cells.