Possible involvement of atypical protein kinase C (PKC) in glucose-sensitive expression of human insulin gene: DNA-binding activity and transcriptional activity of pancreatic and duodenal homeobox gene-1 (PDX-1) are enhancedvia calphostin C-sensitive but phorbol 12-myristate 13-acetate (PMA) and Go 6976-insensitive pathway

Pancreatic and duodenal homeobox gene-1 (PDX-1) is a transcription factor w hich regulates the insulin gene expression. In this study, we tried to eluc idate the role of PDX-1 in the glucose-induced transcriptional activation o f the human insulin gene promoter in MIN6 cells. Electrophoretic mobility s hift assay (EMSA) and chloroamphenicol acetyltransferase (CAT) assay demons trated that both DNA-binding activity and transcriptional activity of PDX-1 were increased with 20 mmol/l glucose more than with 2 mmol/l glucose. The DNA-binding activity of PDX-1 induced by high glucose was blocked by phosp hatase treatment, suggesting the involvement of PDX-1 phosphorylation in th is event. In an in vitro phosphorylation study, PDX-1 was phosphorylated by protein kinase C (PKC), but not by cAMP dependent protein kinase (PKA) or mitogen-activated protein kinase (MAPK). Furthermore, increased PDX-1 funct ion induced by high glucose was blocked by calphotin C, an inhibitor of all PKC isoforms, but unaffected by phorbol 12-myristate 13-acetate (PMA), an activator of classical and novel PKC, or Go 6976, an inhibitor of classical and novel PKC, which suggested that the PKC family which activated PDX-1 i n MIN6 cells was atypical PKC. Western blot and immunocytochemical studies with anti-PKC zeta antibody confirmed the presence of PKC zeta, one of the isoforms of atypical PKC, in MIN6 cells. Furthermore, PKC zeta activity was significantly increased by glucose stimualtion. These results suggest that high glucose increased DNA-binding activity of PDX-1 by activating atypica l PKC including PKC zeta, resulting in transcriptional activation of the hu man insulin gene promoter.