Exp Clin Endocrinol Diabetes 2002; 110(6): 304-309
DOI: 10.1055/s-2002-34594
Article

© Johann Ambrosius Barth

Construction And Characterization of a Conditionally Active Construct of The Insulin-Regulated Forkhead Transcription Factor FKHR

Andreas Barthel 1 , Hans-Martin Orth 1 , Klaus-Dieter Krüger 1 , Dieter Schmoll 2 , Hans-Georg Joost 1
  • 1 Institute of Pharmacology and Toxicology, Technical University of Aachen, Wendlingweg 2, D-52057 Aachen, Germany
  • 2 Department of Medical Biochemistry and Molecular Biology, University of Greifswald, Klinikum, Sauerbruchstrasse, D-17487 Greifswald, Germany
Further Information

Publication History

received 11 March 2002 first decision 23 April 2002

accepted 6 May 2002

Publication Date:
09 October 2002 (online)

Summary

Insulin is known to inhibit glucose-6-phosphatase gene expression through PI 3-kinase/PKB mediated phosphorylation and inactivation of the forkhead transcription factor FKHR, which is a potent transactivator of the glucose-6-phosphatase gene. To study the function and regulation of the transcription factor FKHR in hepatic cells, we constructed a hydroxytamoxifen-inducible version of FKHR by fusing a part of the hormone binding domain of the estrogen receptor (ER) to the C-terminus of FKHR (FKHR-ER). In HepG2-cells transiently transfected with plasmids encoding the FKHR-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. We next generated a H4IIEC3 rat hepatoma cell line stably expressing both FKHR-ER and a glucose-6-phosphatase promoter-based reporter construct. After 2h stimulation with hydroxytamoxifen, the promoter activity was stimulated 3-5 fold, and continued to increase up to 100-fold after 15 h. The response was half maximal at 0.5 μM hydroxytamoxifen. Insulin (1 nM) decreased the hydroxytamoxifen induced promoter activity by about 70% of the maximal response. This cell system can be used for (1) the identification of FKHR dependent genes and for (2) high throughput screening (HTS) of agents affecting the activity of FKHR and its regulation by insulin.

Abbreviations used: FKHR, forkhead in rhabdomyosarcoma; G6Pase, glucose-6-phosphatase; PKB, protein kinase B; PI 3-kinase, phosphatidyl-inositol 3-kinase; IRU, insulin-responsive unit; Tx, 4-hydroxytamoxifen, ER, estrogen receptor; HBD, hormone binding domain

References

Dr. Andreas Barthel

Institute of Pharmacology und Toxicology

RWTH Aachen

Wendlingweg 2

52057 Aachen, Germany

Phone: +49-241-8088642

Fax: +49-241-8082433

Email: [email protected]