Possible Involvement of Atypical Protein Kinase C (PKC) in Glucose-Sensitive Expression of the Human Insulin Gene: DNA-Binding Activity and Transcriptional Activity of Pancreatic and Duodenal Homeobox Gene-1 (PDX-1) Are Enhanced via Calphostin C-Sensitive but Phorbol 12-Myristate 13-Acetate (PMA) and Gö 6976-Insensitive Pathway

Pancreatic and duodenal homeobox gene-1(PDX-1) is a transcription factor which regulates the insulin gene expression. In this study, we tried to elucidate the role of PDX-1 in the glucose-induced transcriptional activation of the human insulin gene promoter in MINE cells. Electrophoretic mobility shift assay (EMSA) and chloramphenicol acetyltransferase (CAT) assay demonstrated that both DNA-binding activity and transcriptional activity of PDX-1 were increased with 20mmol/l glucose more than with 2mmol/l glucose. The DNA-binding activity of PDX-1 induced by high glucose was blocked by phosphatase treatment, suggesting the involvement of PDX-1 phosphorylation in this 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 function induced by high glucose was blocked by calphostin C, an inhibitor of all PKC isoforms, but unaffected by phorbol 12-myristate 13-acetate (PMA), an activator of classical and novel PKC, or Gö 6976, an inhibitor of classical and novel PKC, which suggested that the PKC family which activated PDX-1 in MINE cells was atypical PKC. Western blot and immunocytochemical studies with anti-PKCζ antibody confirmed the presence of PKC ζ, one of the isoforms of atypical PKC, in MIN6 cells. Furthermore, PKC ζ activity was significantly increased by glucose stimulation. These results suggest that high glucose increased DNA-binding activity of PDX-1 by activating atypical PKC including PKC ζ, resulting in transcriptional activation of the human insulin gene promoter.