Involvement of the paired-domain transcription factor Pax6 in the regulation of glucagon gene transcription by insulin
von Rafal Grzeskowiak
Datum der mündl. Prüfung:2000-10-31
Betreuer:Prof. Dr. Willhart Knepel
Gutachter:Prof. Dr. Rüdiger Hardeland
Gutachter:Prof. Dr. Kurt Jungermann
EnglischRegulation of gene transcription is an important aspect of insulin's action. However, the mechanisms involved are poorly understood. Insulin inhibits glucagon gene transcription and insulin deficiency is associated with hyperglucagonemia that contributes to hyperglycemia in diabetes mellitus. In the present study transient transfection analysis in a glucagon-producing pancreatic islet cell line was performed, where the activity of an artificial minienhancer consisting of synergizing Pax6 binding sites (G3A) in front of a heterologous promoter, as well as Pax6 activity when assessed using a GAL4/viral E1B system, were inhibited by insulin. This provides evidence that Pax6 can confer negative regulation by insulin in pancreatic islets. Furthermore, Pax6 seems to play a critical role in the insulin responsiveness of the glucagon promoter because the overexpression of the Pax6 paired domain as well as the mutation of the Pax6 binding sites within the glucagon promoter element G1 and G3 markedly reduced basal activity and insulin responsiveness. The expression of GAL4-Pax6 and GAL4-VP16 restored basal activity of the doubly mutated promoter, whereas only GAL4-Pax6 restored also insulin responsiveness. When the potential Pax6 coactivator CBP was fused to the GAL4 DNA-binding domain, the GAL4-CBP activity was inhibited by insulin within the glucagon promoter context but not in front of the viral E1B promoter. Fusing N-, C-terminal and middle parts of CBP with the GAL4 domain only the N- and C-terminal part conferred transcriptional activity and insulin responsiveness. Further mapping of the C-terminal of CBP revealed a single region between amino acids 2040 and 2170 sufficient to confer the negative regulation by insulin. The activity conferred by this region as well as by the N-terminal part of CBP was inhibited by the overexpression of the constitutively active form of PKB, myrPKB. The results of present study suggest that the paired domain transcription factor Pax6 is required for insulin responsiveness of the glucagon promoter. They alsoindicate that the Pax6-interacting coactivator CBP might be involved in this process. It is finally speculated that within the specific context of the glucagon promoter a nucleoprotein complex is being induced with Pax6 and CBP as critical components. Insulin-induced signalling pathways might target this large, glucagon promoter-specific protein complex rather than any single transcriptional factor and therefore act through a IRE-binding factor (IRF)-independent mechanism.
Keywords: Gene regulation; Insulin; glucagon; Pax6; CBP; cell signalling; transcription