|Knepel, Willhart Prof. Dr.
|Involvement of the paired-domain transcription factor Pax6 in the regulation of glucagon gene transcription by insulin
|Hardeland, Rüdiger Prof. Dr.
|Regulation 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.
|Jungermann, Kurt Prof. Dr.
|mathematics and natural science