| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Pancreatic and duodenal homeobox gene-1 (PDX-1) is a transcription factor which regulates the insulin gene expression. Previously, we reported 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 involvement of PDX-1 phosphorylation in this event, and PDX-1 was phosphorylated by protein kinase C (PKC) in in vitro. We then tried to elucidate the more detailed mechanism of PDX-1 in the glucose-induced transcriptional activation of the human insulin gene promoter in MIN6 cells. Increased PDX-1 function induced by high glucose was blocked by Calphostin C, an inhibitor of all PKC isoforms, but unaffected by Go6976, an inhibitor of classical and novel PKC, which suggested that the PKC family which activated PDX-1 in MIN6 cells was atypical PKC.Western blot and immunocytochemical studies with anti-PKCζ antibody confirmed the presence of PKCζ, one of the isoforms of atypical PKC, in MIN6 cells. Furthermore, PKCζ 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 PKCζ, resulting in transcriptional activation of the human insulin gene promoter. To confirm the role of PKCζ in the activation of PDX-1, we tried to study the effect of the dominant negative PKCζin MIN6 cells, and we could confirm the transfected dominant negative PKCζ protein by Western blot.
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