Herstellung und Charakterisierung einer induzierbaren Form des Transkriptionsfaktors FOXO1a

In order to characterize biological functions mediated by the insulin-regulated transcription factor FOXO1a, a 4-hydroxytamoxifen-inducible version of FOXO1a was constructed by fusing a part of the hormone-binding domain of the estrogen receptor (ER) to the C-terminus of FOXO1a (FOXO1a-ER). In HEP-G2-cells transiently transfected with plasmids encoding the FOXO1a-ER fusion protein and a glucose-6-phosphatase reporter construct, hydroxytamoxifen induced a marked induction of glucose-6-phosphatase promoter activity, whereas no effect was observed in control cells. Subsequently, by use of a retroviral transfection system, a H4 rat hepatoma cell line stably expressing both FOXO1a-ER and a glucose-6-phosphatase promoter-based reporter construct was generated. After two hours stimulation with hydrotamoxifen, the promoter activity was stimulated three- to five-fold and continued to increase up to 100-fold after 15 hours. The response was half maximal at 0.5µM hydroxytamoxifen. Insulin (1nM) decreased the hydroxytamoxifen induced promoter activity by about 70% of the maximal response. Furthermore, the transactivation of the glucose-6-phosphatase gene by the FOXO1a-ER construct could be shown to influence expression of mRNA and its subsequent protein in the H4 cell line. Following stimulation of the glucose-6-phosphatase activity with dexamethasone/cAMP and activation of the FOXO1a construct a functional resistence towards insulin was shown on the level of mRNA, which highlights the possible prodiabetogenic effect of FOXO1a. The forementioned cell system can theoretically be used for the identification of other FOXO1a-dependently regulated genes and for the identification of agents affecting the activity of FOXO1a and its regulation by insulin.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2012

Author dc:creator

• Orth, Hans-Martin

Contributors dc:contributor

• Barthel, Andreas

• info:eu-repo/classification/ddc/610
• Transkriptionsfaktor
• Diabetes mellitus
• Insulin
• Glucosephosphatase <Glucose-6-Phosphatase>
• Medizin
• FOXO1a

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113406%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51089